CN114894865A - A kind of high-precision glucose sensor and preparation method thereof - Google Patents

Abstract

The invention discloses a high-precision glucose sensor and a preparation method thereof. The method comprises the following steps: (1) activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of a working electrode; (2) and (3) coating the enzyme solution containing the glucose oxidase on a reaction area of the working electrode, crosslinking at room temperature, and then cleaning and drying. The biosensor prepared by the invention has the service life of 14 days at 4 ℃, and has reversible electrochemical characteristics, low working potential and strong anti-interference capability.

Description

A kind of high-precision glucose sensor and preparation method thereof

technical field

The invention belongs to the technical field of biosensors, and in particular relates to a high-precision glucose sensor and a preparation method thereof.

Background technique

Diabetes has become one of the diseases that threaten human health. Blood sugar monitoring in diabetics is very important to control the condition. Patients are often afraid and uncomfortable with invasive tests that are now commonly used. Therefore, the research and development of non-invasive blood glucose meters has become a key topic in the medical field of various countries. Among them, the reverse iontophoresis technology was used to obtain the sugar molecules in the subcutaneous interstitium, and the glucose exuded from the subcutaneous tissue was tested by the electrochemical biosensor. Related research has been carried out abroad. Compared with direct blood sampling to detect human blood sugar (in the order of mmol/L), the concentration of subcutaneously exuded glucose is very low (in the order of μmol/L), and traditional blood glucose biosensors can hardly meet the requirements in terms of detection range and sensitivity. However, sensors that can detect low-concentration glucose cannot be practical due to the limitation of electrode fabrication. The non-invasive detection of subcutaneous blood glucose was achieved by using a hydrogen peroxide sensor at a working voltage of 0.42V. However, the higher working potential easily causes other electroactive substances in the body fluid to easily interfere with the test.

To avoid the disadvantages of enzymes, enzyme-free glucose sensors have received increasing attention. Precious metals, such as platinum, gold, and palladium, have been used in the development of enzyme-free glucose sensors due to their stable oxidative catalytic activity towards glucose. However, the catalytic kinetics of glucose oxidation is poor, the reaction time is long, the sensitivity is low, and the reaction intermediates and ^Cl- in the electrolyte are toxic to noble metal electrodes. In addition, the electrochemical potential of glucose oxidation is usually high enough to catalyze some of the body's own physiological substances, resulting in poor anti-interference ability.

In order to improve sensitivity and selectivity, surface modification techniques such as electroplating, anodization, and vapor deposition have been studied to modify the surface of sensing electrodes. These techniques can increase the surface roughness of the electrode, accelerate the transfer of electrons from glucose to the electrode, and improve the sensitivity of the sensor. Furthermore, on noble metal electrodes, the oxidation of common physiological interfering substances such as ascorbic acid (AA) and uric acid (UA) is a diffusion-controlled process, whereas glucose oxidation is controlled by surface reactions. The increase in surface roughness reduces the diffusion of interfering species but does not affect the reaction rate of glucose, thereby increasing the selectivity. Although it is theoretically possible to improve the selectivity and sensitivity of the sensor by increasing the electrode surface roughness, in practice, the performance improvement of this method is limited.

SUMMARY OF THE INVENTION

In view of the above deficiencies in the prior art, the present invention provides a high-precision glucose sensor and a preparation method thereof.

For realizing the above-mentioned purpose, the technical scheme that the present invention solves its technical problem adopts is:

A preparation method of a high-precision glucose sensor, comprising the following steps:

(1) Activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of the working electrode;

(2) Coat the enzyme solution containing glucose oxidase on the reaction zone of the working electrode, wash and dry after cross-linking at room temperature.

Further, the three-electrode system consists of a glassy carbon working electrode, a glassy carbon counter electrode and an Ag/AgCl reference electrode.

Further, the process of activation treatment is:

The electrode surface can be cleaned with 50% nitric acid solution, absolute ethanol and ultrapure water in sequence.

Further, the osmium redox polymer coupled with chitosan was adsorbed to the surface of the working electrode under the condition of avoiding light at room temperature.

Further, the glucose oxidase and osmium are cross-linked by glutaraldehyde in a vacuum environment for 3-5 days to form an osmium redox polymer, wherein the mass concentration of glutaraldehyde is 2-5%.

Further, the concentration of glucose oxidase in the enzyme solution is 2-5 U/μL, and the addition amount of BSA is 1.5-2% of the volume of the enzyme solution.

Further, the cross-linking agent used for cross-linking in step (2) is glutaraldehyde, and the addition amount of the cross-linking agent in the enzyme solution is 2-5% of the volume of the enzyme solution.

Further, the cross-linking time in step (2) is 3-5h.

Further, the area of the sensitive part of the working electrode is 0.85 cm.

The high-precision glucose sensor prepared by the above method.

When the sensor prepared by the invention is used, two sensors with the same shape cooperate together to complete the extraction and detection functions of the sensor. Among them, W1 and W2 are thin-film glassy carbon working electrodes, C1 and C2 are glassy carbon counter electrodes, W and R form an extraction electrode circuit, and R1 and R2 are screen-printed Ag/AgCl reference electrodes, which are used for sensor testing. a stable reference voltage. (see picture 1)

Beneficial effects of the present invention:

1. The present invention uses the osmium redox polymer coupled with chitosan as the electron mediator to modify the thin-film glassy carbon electrode, so that the osmium redox polymer has reversible electrochemical properties; A rapid transfer of electrons takes place, eliminating the dependence on the oxygen concentration in the bulk solution.

2. The present invention adopts the glutaraldehyde method to immobilize glucose enzyme molecules to prepare a new type of biosensor, which effectively reduces the working voltage of the sensor; at the same time, the cross-linked enzyme solution has a low working potential, which can significantly improve its anti-interference ability.

3. The material preparation methods adopted in the present invention are all carried out in an aqueous solution without using organic solvents, the production process is green and environmentally friendly, and at the same time, there is no organic solvent residue, which is conducive to expanding the scope of application.

4. The present invention has cheap raw materials and simple synthesis path, and can quickly and massively prepare conductive materials, which is beneficial to the mass production and commercial application of new flexible biological electronic devices.

5. The sensor prepared by the present invention has a sensitivity of 250.55nA/(μmol·L ^-1 ), a minimum detection limit of 0.018μmol/L, and a correlation coefficient of 0.999 within the standard glucose concentration range of 5.0-25.0mmol/L; In the standard subcutaneous glucose concentration range of 5.0-30.0mmol/L, the extracted glucose current response value has a linear relationship with the subcutaneous glucose concentration, the linear correlation coefficient is 0.993, and the sensitivity is 343.21nA/(mmol·L ^-1 ) ; The accuracy of a single sensor for 10 mmol/L glucose detection is 1.34% (n=10), and the accuracy of 20 mmol/L glucose measurement between different sensors is 2.22% (n=10). Lifespan up to 14 days.

Description of drawings

Figure 1 is a three-electrode architecture diagram of the sensor;

Figure 2 is a graph of the i-t response of the sensor to different glucose concentrations;

Figure 3 is a linear graph of the sensor against different glucose concentrations;

Figure 4 is a graph of the long-term stability of the sensor.

Detailed ways

The specific embodiments of the present invention are described below to facilitate those skilled in the art to understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes Such changes are obvious within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the inventive concept are within the scope of protection.

Example 1

A preparation method of a high-precision glucose sensor, the specific process is as follows:

(1) Using glassy carbon working electrode, glassy carbon counter electrode and Ag/AgCl reference electrode to form a three-electrode system; wherein, the area of the working electrode reaction zone is 0.85cm;

(2) sequentially using 50% nitric acid solution, absolute ethanol and ultrapure water to clean the electrode surface to complete the activation treatment of the electrode;

(3) Using glutaraldehyde as a cross-linking agent, the glucose oxidase and osmium were cross-linked in a vacuum environment for 3 days to form an osmium redox polymer, and then the chitosan was coupled with the osmium redox polymer, and absorbed 6 μL of the coupled product was added dropwise to the surface of the working electrode, and kept overnight at room temperature in the dark, so that the polymer was firmly adsorbed on the surface of the working electrode;

(4) Draw 20 μL of enzyme solution and evenly apply it to the reaction area of the working electrode. The content of glucose oxidase in the enzyme solution is 2U/μL, the weight of BSA is 1.5% of the volume of the enzyme solution, and the content of glutaraldehyde is 1.5% of the volume of the enzyme solution. 2.7%, then cross-linked at room temperature for 3h;

(5) Rinse the product obtained in step (4) with deionization and PBS to remove free substances. After drying, the product is stored in a refrigerator at 4° C. for future use.

Example 2

A preparation method of a high-precision glucose sensor, the specific process is as follows:

(1) Using glassy carbon working electrode, glassy carbon counter electrode and Ag/AgCl reference electrode to form a three-electrode system; wherein, the area of the working electrode reaction zone is 0.85cm;

(2) sequentially using 50% nitric acid solution, absolute ethanol and ultrapure water to clean the electrode surface to complete the activation treatment of the electrode;

(3) Using glutaraldehyde as a cross-linking agent, the glucose oxidase and osmium were cross-linked in a vacuum environment for 3 days to form an osmium redox polymer, and then the chitosan was coupled with the osmium redox polymer, and absorbed 6 μL of the coupled product was added dropwise to the surface of the working electrode, and kept overnight at room temperature in the dark, so that the polymer was firmly adsorbed on the surface of the working electrode;

(4) Draw 20 μL of enzyme solution and evenly apply it to the reaction zone of the working electrode. The content of glucose oxidase in the enzyme solution is 3 U/μL, the weight of BSA is 2% of the volume of the enzyme solution, and the content of glutaraldehyde is 3 U/μL of the volume of the enzyme solution. 2.5%, then cross-linked at room temperature for 3h;

(5) Rinse the product obtained in step (4) with deionization and PBS to remove free substances. After drying, the product is stored in a refrigerator at 4° C. for future use.

Example 3

A preparation method of a high-precision glucose sensor, the specific process is as follows:

(1) Using glassy carbon working electrode, glassy carbon counter electrode and Ag/AgCl reference electrode to form a three-electrode system; wherein, the area of the working electrode reaction zone is 0.85cm;

(2) sequentially using 50% nitric acid solution, absolute ethanol and ultrapure water to clean the electrode surface to complete the activation treatment of the electrode;

(3) Using glutaraldehyde as a cross-linking agent, the glucose oxidase and osmium were cross-linked in a vacuum environment for 3 days to form an osmium redox polymer, and then the chitosan was coupled with the osmium redox polymer, and absorbed 6 μL of the coupled product was added dropwise to the surface of the working electrode, and kept overnight at room temperature in the dark, so that the polymer was firmly adsorbed on the surface of the working electrode;

(4) Draw 20 μL of enzyme solution and evenly apply it to the reaction zone of the working electrode. The content of glucose oxidase in the enzyme solution is 4 U/μL, the weight of BSA is 1.8% of the volume of the enzyme solution, and the content of glutaraldehyde is 1.8% of the volume of the enzyme solution. 3%, and then cross-linked at room temperature for 3h;

(5) Rinse the product obtained in step (4) with deionization and PBS to remove free substances. After drying, the product is stored in a refrigerator at 4° C. for future use.

Experimental example

1. Electrochemical properties of the working electrode

8 μL of osmium polymer modified thin-film glassy carbon working electrode was used. At different scan rates, the changes of the cathodic and anodic peak currents of the redox polymers increased with the scan rate. The scan rates are 5, 10, 20, 50, 100, 200, 500mV/s: the ratio of the peak current (I _pa ) to the peak current (I _pc ) of the reduction is I _pa /I _pc ≈ 1, and the square root of the peak current and scan rate The relationship is linear, indicating that the osmium redox polymer has reversible electrochemical properties.

2. Detection of the current response of the sensor to standard glucose

Figures 2 and 3 show the current response curves of the sensor to standard glucose. Each sample was measured 3 times, and the working voltage was 0.21V. It can be seen from the figure that when the glucose solution is added, the current response of the sensor also increases gradually with the increase of the glucose concentration. Meanwhile, in the concentration range of 5.0-25mmol/L, the current response signal of glucose solution has a very good linear relationship. The linear correction equation of the sensor in the range of 5.0-25mmol/L I _p =0.184x-9.44, the lowest detection limit is 0.018μmol/L, the correlation coefficient R is 0.99937, and the sensitivity of the sensor is 343.21nA/(μmol·L ^-1 ) . The results show that the use of osmium polymer effectively reduces the operating voltage of the sensor.

3. Sensor current response to subcutaneous glucose

The experiment of percutaneous extraction and detection of subcutaneous glucose was carried out by reverse iontophoresis. The extraction process of subcutaneous glucose is a conventional process in the art, and the specific process can refer to the published literature. Among them, the concentration range of subcutaneous glucose is 0, 3, 7, 10, 14, 19 mmol/L respectively, the sensor is attached to the surface of the mouse skin, the extraction and detection period of the sensor is 10 min, the working voltage is 0.21 V, and the current response value of the sensor for 100 s is taken as Test results. The sensor meets the current of the extracted glucose and the reverse iontophoresis technology of subcutaneous glucose to carry out the transdermal extraction and detection experiment of subcutaneous glucose. The results show that the current encounter of the sensor on the extracted glucose is not much different from that of the subcutaneous glucose.

4. Sensor repeatability detection

The accuracy of the developed single biosensor in the same batch and different sensors between batches of 10 mmol/L glucose repeated tests were studied. The current response results of the sensor were obtained by repeated testing of 10 mmol/L glucose for 10 times at a response time of 100 s. The intra-assay accuracy refers to the current response results of a single sensor repeating 10 tests of 10 mmol/L glucose. The batch-to-batch accuracy refers to the current response results of 10 sensors testing 10 mmol/L glucose respectively. As shown in Fig. 4, the intra- and inter-assay accuracies of the sensor are shown to be 4.07% and 3.22%, respectively. All are less than 5%, indicating that the prepared sensor has good repeatability and consistency.

5. Sensor stability detection

The traditional life test is to evaluate the performance of the sensor by measuring how long it is stored at 4°C. In physical chemistry, the rate of chemical reactions increases as the reaction temperature increases. Biological reagents are stored at different temperatures, and the "rate" of reagent failure and the temperature and time of storage are also in line with the physicochemical reaction rate relationship. Arrhenius empirically summed up this law of physical chemistry. Arrhenius described the empirical formula for the effect of temperature on the reaction rate as follows: lnk=-E _a RT+B, where k is the reaction rate; T is the absolute temperature of the reaction. Design experiments to carry out the storage time for the failure of the storage reagent at multiple temperatures, and the reciprocal of this time (days or hours) is the rate at which the reagent deteriorates at this temperature. Convert the test temperature to absolute temperature T and the reciprocal storage time to form each data and plot it on semi-logarithmic paper, let lnk be Y and 1/T be X. A line of best fit was drawn on the graph, and the line was backtracked to the logarithmic value of the reaction rate when T was 277K (4°C). The antilog (exponent) is obtained from the logarithmic value, and then the reciprocal of the value is obtained, which is the estimated stabilization time. In this paper, the life-stable performance of glucose biosensors was observed based on the above principles. Tests were carried out at 20, 35, 55°C. Taking the response of the biosensor at 4°C as the test result for the control product, the test result is 90% or less of the response result of the biosensor at 4°C as the failure.

Plot the paired results of X(1/T) and Y[lg(1/d)] or perform a linear regression process (linear relationship on semi-logarithmic paper). It is estimated from the regression formula that under the absolute temperature of 277K at 4°C, the expected stable days is 14d. Therefore, it is estimated from the experiment that the prepared biosensor can be stored stably for about 14 days at 4°C.

Claims (9)

1. A preparation method of a high-precision glucose sensor is characterized by comprising the following steps:

(1) activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of a working electrode;

(2) and (3) coating the enzyme solution containing the glucose oxidase on a reaction area of the working electrode, crosslinking and fixing at room temperature, and then cleaning and drying.

2. The method of claim 1, wherein the three-electrode system consists of a glassy carbon working electrode, a glassy carbon counter electrode, and an Ag/AgCl reference electrode.

3. The method according to claim 1, wherein the activation treatment is performed by:

and cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water.

4. The method of claim 1, wherein the osmium redox polymer to which chitosan is coupled is adsorbed to the surface of the working electrode at room temperature in the absence of light.

5. The method of claim 1 or 4, wherein the osmium redox polymer is formed by cross-linking glucose oxidase with osmium via glutaraldehyde.

6. The method according to claim 1, wherein the concentration of glucose oxidase in the enzyme solution is 2 to 5U/. mu.L, and the amount of BSA added is 1.5 to 2% by volume of the enzyme solution.

7. The preparation method according to claim 1, wherein the crosslinking agent used in the step (2) is glutaraldehyde, and the addition amount of the glutaraldehyde in the enzyme solution is 2-5% by volume of the enzyme solution.

8. The method according to claim 1, wherein the crosslinking time in step (2) is 3 to 5 hours.

9. A high-precision glucose sensor produced by the method according to any one of claims 1 to 8.

Images